1. Field of the Invention
The present invention relates to methods for the selection of antibodies which bind to selected epitopes utilizing phage display of antibody combinatorial libraries. The invention also relates to antibodies prepared by such methods.
2. Related Art
In the postgenomic era, efforts in drug development can now be focused on finding methods to specifically block the function of key proteins previously identified by such techniques as microarray analysis of mRNA expression levels in disease states. Proteomics is the new science encompassing understanding the way proteins interact with each other both in coordinated pathways and as binding partners. Structure activity relationships for proteins include the mapping of common domains and identifying three-dimensional conformations responsible for functions. Access to three-dimensional (3D) information on proteins has also become routine. For example the NCBI maintains public access to a tool called VAST which is a structure-structure similarity search service. It compares 3D coordinates of a newly determined protein structure to those in the molecular modeling database (MMDB) and the protein database (PDB).
Phage display technology describes an in vitro selection technique in which the polynucleotide sequence encoding a peptide or protein is genetically fused to a coat protein of a bacteriophage, resulting in display of the fused protein on the exterior of the phage virion, while the DNA encoding the fusion resides within the virion. This physical linkage between the displayed protein and the DNA encoding it allows screening of vast numbers of variants of the protein, each linked to its corresponding DNA sequence, by a simple in vitro selection procedure called “biopanning”.
Phage, ribosome, yeast, and bacterial display libraries are tools for querying large numbers of proteins or peptides. Ribosome display is a method of translating mRNAs into their cognate proteins while keeping the protein attached to the RNA. The nucleic acid coding sequence is recovered by RT-PCR (Mattheakis, L. C. et al. 1994. Proc. Natl. Acad. Sci. USA 91, 9022). Yeast display is based on the construction of fusion proteins of the membrane-associated alpha-agglutinin yeast adhesion receptor, aga1 and aga2, a part of the mating type system (Broder, et al. 1997. Nature Biotechnology, 15:553-7). Bacterial display is based on fusion of the target to exported bacterial proteins that associate with the cell membrane or cell wall (Chen and Georgiou. 2002. Biotechnol Bioeng, 79:496-503).
As compared to hybridoma technology, phage and other antibody display methods afford the opportunity to manipulate selection against the antigen target in vitro and without the limitation of the possibility of host effects on the antigen or vice versa. One particular advantage of in vitro selection methods is the ability to manipulate selection procedures to obtain antibodies binding to diverse sites on the target protein.
While phage libraries simplify the retrieval of genetic material associated with functional attributes, multistep panning strategies are required to isolate the best candidate from the library. On the other hand, in those instances where structural information concerning the functional domain of a polypeptide ligand is known, it would be desirable to have a method to select antibodies or other binding partners such as peptides or proteins which bind to a ligand at specific defined domains. Domain or epitope directed pannings have become a routine way of selecting antibodies that bind to a target protein. Such selections have primarily been achieved by employing a stepwise selection of antibodies utilizing methods known variously as selective panning, de-selective panning, ligand capture, subtractive panning or pathfinder selection (Hoogenboom, H. R. et al (2000) supra).
In subtractive panning, target(s) with overlapping but not completely identical binding sites can be used to de-select unwanted binders. This strategy has been used to identify binders even to unknown antigens as in the use of normal cells to de-select binders to cancer cells. Alternatively, naturally occurring proteins with some common domains or structure are used in sequential or competition selection to obtain antibodies binding to sites that differ or are common among the related antigens. Typically, these studies have utilized naturally occurring proteins such as related chemokines or mutant H-ras proteins (Horn, I. R. et al. 1999, FEBS Lett. 463:115-120).
Ligand-capture directed panning is analogous to an ELISA sandwich assay in that an immobilized antibody to an irrelevant and non-adjacent epitope is used to capture and present the preferred binding face of the target ligand for phage panning (U.S. Pat. No. 6,376,170). Others have used competing antibodies to selectively mask the antigen at other than the desired target domain (Tsui, P. et al. 2002. J. Immunol. Meth. 263:123-132). Pathfinder technology uses monoclonal and polyclonal antibodies, as well as natural ligands conjugated directly or indirectly to horseradish peroxidase (HRP). In the presence of biotin tyramine these molecules catalyze biotinylation of phage binding in close proximity to the target antigen, allowing specific recovery of ‘tagged’ phage from the total population using streptavidin. In this way, phage binding to the target itself, or in its immediate proximity, are selectively recovered (Osborn, J. K. et al. 1998. Immunotechnol. 3: 293-302). The use of monoclonal antibodies to direct binding to alternate sites has also been termed “epitope walking” (Osborn, J. K. et al. 1998. supra).
These methods suffer from the drawback that an entire effort directed to obtaining and characterizing an undesirable binding partner must precede the effort to obtain a binding partner to the desired domain and that a specific epitope is not targeted. The present invention provides a novel method to obtain antibodies or ligand binding partners that bind to a selected epitope by incorporating a hybrid competitor protein into the panning selection process.